1. Field of the Invention
The present invention relates generally to liposomes, and more particularly to a method of producing liposomes useful for encapsulating biologically active materials. The liposomes are, therefore, useful in applications such as in vivo drug delivery and gene therapy and as diagnostic agents.
2. Description of the Background
Liposomes are microscopic vesicles, generally spherically shaped, formed from one or more lipid walls. The walls are prepared from lipid molecules, which have the tendency both to form bilayers and to minimize their surface area. The lipid molecules that make up a liposome have hydrophilic and lipophilic portions. Upon exposure to water, the lipid molecules form a bilayer membrane wherein the lipid ends of the molecules in each layer are directed to the center of the membrane, and the opposing polar ends form the respective inner and outer surfaces of the bilayer membrane. Thus, each side of the membrane presents a hydrophilic surface while the interior of the membrane comprises a lipophilic medium.
Liposomes can be classified into several categories based on their overall size and the nature of the lamellar structure. The classifications include small unilamellar vesicles (SUV), multilamellar vesicles (MLV), large unilamellar vesicles (LUV), and oligolamellar vesicles. SUVs range in diameter from approximately twenty to fifty nanometers and consist of a single lipid bilayer surrounding an aqueous compartment. A characteristic of SUVs is that a large amount of the total lipid, about 70%, is located in the outer layer of the bilayer. The most frequently encountered and easily prepared liposomes are the multilamellar vesicles. Where SUVs are single compartment vesicles of a fairly uniform size, MLVs vary greatly in diameter up to about 30,000 nanometers and are multicompartmental in their structure wherein the liposome bilayers are typically organized as closed concentric lamellae with an aqueous layer separating each lamella from the next. Large unilamellar vesicles are so named because of their large diameter which ranges from about 600 nanometers to 30 microns. Oligolamellar vesicles are intermediate liposomes having a larger aqueous space than MLVs and a smaller aqueous space than LUVs. Oligolamellar vesicles have more than one internal compartment and possibly several concentric lamellae, but they have fewer lamellae than MLVs.
A variety of methods for preparing liposomes are known in the art, several of which are described in Liposome Technology (Gregoriadis, G., editor, three volumes, CRC Press, Boca Raton 1984) or have been described by Lichtenberg and Barenholz in Methods of Biochemical Analysis, Volume 33, 337-462 (1988). Liposomes are also well recognized as useful for encapsulating biologically active materials. Preparation methods particularly involving the encapsulation of DNA by liposomes, and methods that have a direct application to liposome-mediated transfection, have been described by Hug and Sleight in Biochimica and Biophysica Acta, 1097, 1-17 (1991).
When liposomes form, solvent and solute molecules become trapped in their lumen. The volume encapsulated, or capture volume, is dependent on the size of the liposomes, the lipid composition of the vesicles and the ionic composition of the medium. The fraction of the solvent entrapped by liposomes is defined as the encapsulation or entrapment efficiency and is proportional to both lipid concentration and vesicle radius. The fraction of solute sequestered inside the liposomes is generally directly proportional to the fraction of solvent entrapped.
Although the encapsulation of biologically active materials in liposomes has significant potential for delivering such materials to targeted sights in the human body, the production of encapsulated materials on a commercially feasible scale has been a problem. In order for liposomes to be used more widely for therapeutic purposes, it is desirable that the preparation process satisfy the following standards:
1) a high degree of encapsulation can be attained;
2) organic solvent or detergent can be completely removed from the final product or their use avoided;
3) the final product is obtainable by a simple procedure;
4) preparation can be carried out on a large scale;
5) the stability of the liposomes supports an appropriate storage period; and
6) the encapsulated material is not partially or completely denatured or inactivated during liposome production or encapsulation.
A method for preparing liposomes with a water-soluble, biologically active compound using lyophilization is disclosed in U.S. Pat. No. 4,311,712 to Evans, et. al. Evans, et. al., however, state that their methods are not particularly suitable for aqueous soluble materials. Moreover, the disclosed method of preparation requires the mixture of the biologically active material with an organic solvent. Felgner, et al. (EP 172 007 B) also describe a technique incorporating an organic solvent. One object of the present invention is to avoid the use of organic solvents or detergents in forming the liposomes because these substances are difficult to remove, present health hazards or interact unfavorably with the biologically active molecules to be encapsulated.
It is also known that liposomes and their contents may be relatively unstable in aqueous dispersion. Accordingly, an attempt to increase the relatively short storage life of certain liposomal products by dehydrating the dispersion has been the focus of several liposome preparation methods. For example, the aqueous dispersion of encapsulated material is lyophilized to form a stable powder which can be stored for a long period and from which, with an aqueous medium, a liposome dispersion can be reconstituted (see Schneider, et. al. in U.S. Pat. No. 4,229,360). U.S. Pat. No. 4,515,736 (D. Deamer) also describes an encapsulation method in which liposome dispersions are dried in the presence of the material to be encapsulated. As the solution is dried to a highly viscous concentrated mixture, the individual liposomes fuse to form multilamellar structures which capture the material to be encapsulated between the lipid lamellae. Upon rehydration, lipid vesicles form which encapsulate the material. Crowe, et al. (U.S. Pat. No. 4,857,319) describe a method for preserving liposomes involving freeze drying a mixture containing the lipid vesicles, the material to be encapsulated and a disaccharide preserving agent. Each of these methods requires that the biologically active material be subjected to the lyophilization procedure. In contrast, it is a further object of the present invention to encapsulate a material while avoiding the need to subject that material to such rigorous manipulations as lyophilization, thereby decreasing the possibility of physically inactivating or degrading the material to be encapsulated.
Mayer, et. al. (U.S. Pat. No. 5,077,056) describe a liposome preparation method involving the use of small ions to produce gradients which enhance the retention of charged biologically active agents, and optionally involving subjecting the liposome and biologically active agents to a freeze-thaw process during the encapsulation procedure. The method (as further described by Mayer, et al in Biochimica Biophysica Acta 817: 193-196; 1985) produces xe2x80x9cfreeze and thaw multilamellar vesiclesxe2x80x9d (FATMLV). The FATMLV method requires that freezing and thawing be done in the presence of the material to be entrapped. In addition, while high capture of small ions (22Na+ and Mg2+) was demonstrated using the FATMLV method, no encapsulation of macromolecules was achieved. It is yet a further object of the present invention to avoid the need of subjecting the material to be encapsulated to such harsh physical manipulations, and thereby reduce the possibility of inactivating or degrading that material.
It is also an object of the present invention to provide a suitable method for encapsulating a wide variety of biologically active materials including, but not limited to, foods or nutritional substances as well as pharmaceutical agents, DNA, RNA, mRNA, nucleic acids, proteins, polypeptides, peptides and enzymes. It is a further object of the present invention to provide a method which is simple, feasible and inexpensive for the large-scale commercial production of liposomes and encapsulated materials.
The present invention includes a novel method for preparing liposomes and encapsulating a biologically active material therein. The method involves: hydrating and mixing a quantity of lipid in an aqueous solution to form a liposome dispersion in the absence of an organic solvent or detergent; subjecting the liposome dispersion to one or more cycles of freezing and thawing; and dehydrating the liposome dispersion to form a lipid powder. The lipid powder is suitable for both long-term storage and reconstitution to form liposomes and to encapsulate a biologically active material. The lipid powder is hydrated in the presence of the biologically active material whereby the material is encapsulated in reconstituted liposomes. The method may optionally include the step of microfluidizing the reconstituted liposomes and/or the step of separating encapsulated biologically active material from unencapsulated material.
The lipid can be a single or a combination of synthetic and natural lipid molecules. In addition, the method can include combining the liposome dispersion with a bulking agent prior to dehydration and formation of the lipid powder. The weight to weight ratio of such a bulking agent to lipid is approximately 0.1:1 to 2:1.
The present invention provides a novel method for encapsulating a wide variety of biologically active materials. There is no need to subject the material to be encapsulated to harsh physical manipulations, and therefore, the possibility of inactivating or degrading that material is reduced. Moreover, the use of organic solvents or detergents in forming the liposomes is advantageously avoided.